Anti-APOBEC3G activity of HIV-1 Vif protein is attenuated in elite controllers.

UNLABELLED: HIV-1-infected individuals who control viremia to below the limit of detection without antiviral therapy have been termed elite controllers (EC). Functional attenuation of some HIV-1 proteins has been reported in EC. The HIV-1 accessory protein Vif (virion infectivity factor) enhances viral infectivity through anti-retroviral factor apolipoprotein B mRNA editing enzyme catalytic polypeptide-like 3G (APOBEC3G) degradation; however, little is known regarding Vif function in EC. Here, the anti-APOBEC3G activities of clonal, plasma HIV RNA-derived Vif sequences from 46 EC, 46 noncontrollers (NC), and 44 individuals with acute infection (AI) were compared. Vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped viruses were generated by cotransfecting 293T cells with expression plasmids encoding patient-derived Vif, human APOBEC3G, VSV-G, and a vif/env-deficient luciferase-reporter HIV-1 proviral DNA clone. Viral stocks were used to infect 293T cells, and Vif anti-APOBEC3G activity was quantified in terms of luciferase signal. On average, the anti-APOBEC3G activities of EC-derived Vif sequences (median log10 relative light units [RLU], 4.54 [interquartile range {IQR}, 4.30 to 4.66]) were significantly lower than those of sequences derived from NC (4.75 [4.60 to 4.92], P < 0.0001) and AI (4.74 [4.62 to 4.94], P < 0.0001). Reduced Vif activities were not associated with particular HLA class I alleles expressed by the host. Vif functional motifs were highly conserved in all patient groups. No single viral polymorphism could explain the reduced anti-APOBEC3G activity of EC-derived Vif, suggesting that various combinations of minor polymorphisms may underlie these effects. These results further support the idea of relative attenuation of viral protein function in EC-derived HIV sequences. IMPORTANCE: HIV-1 elite controllers (EC) are rare individuals who are able to control plasma viremia to undetectable levels without antiretroviral therapy. Understanding the pathogenesis and mechanisms underpinning this rare phenotype may provide important insights for HIV vaccine design. The EC phenotype is associated with beneficial host immunogenetic factors (such as HLA-B*57) as well as with functions of attenuated viral proteins (e.g., Gag, Pol, and Nef). In this study, we demonstrated that HIV-1 Vif sequences isolated from EC display relative impairments in their ability to counteract the APOBEC3G host restriction factor compared to Vif sequences from normal progressors and acutely infected individuals. This result extends the growing body of evidence demonstrating attenuated HIV-1 protein function in EC and, in particular, supports the idea of the relevance of viral factors in contributing to this rare HIV-1 phenotype.

APOBEC3G oligomerization is associated with the inhibition of both Alu and LINE-1 retrotransposition.

Alu and LINE-1 (L1), which constitute ~11% and ~17% of the human genome, respectively, are transposable non-LTR retroelements. They transpose not only in germ cells but also in somatic cells, occasionally causing cancer. We have previously demonstrated that antiretroviral restriction factors, human APOBEC3 (hA3) proteins (A-H), differentially inhibit L1 retrotransposition. In this present study, we found that hA3 members also restrict Alu retrotransposition at differential levels that correlate with those observed previously for L1 inhibition. Through deletion analyses based on the best-characterized hA3 member human APOBEC3G (hA3G), its N-terminal 30 amino acids were required for its inhibitory activity against Alu retrotransposition. The inhibitory effect of hA3G on Alu retrotransposition was associated with its oligomerization that was affected by the deletion of its N-terminal 30 amino acids. Through structural modeling, the amino acids 24 to 28 of hA3G were predicted to be located at the interface of the dimer. The mutation of these residues resulted in abrogated hA3G oligomerization, and consistently abolished the inhibitory activity of hA3G against Alu retrotransposition. Importantly, the anti-L1 activity of hA3G was also associated with hA3G oligomerization. These results suggest that the inhibitory activities of hA3G against Alu and L1 retrotransposition might involve a common mechanism.

Membrane-associated RING-CH (MARCH) 8 mediates the ubiquitination and lysosomal degradation of the transferrin receptor.

The transferrin receptor (TfR) mediates the uptake of transferrin (Tf)-bound iron from the plasma into the cells of peripheral tissues. The TfR continuously recycles between the plasma membrane and early/recycling endosomes. TfR expression is tightly controlled by the intracellular iron concentration through the regulation of TfR mRNA stability. However, much less is known about the mechanism by which TfR is degraded in cells. Previously, we reported a correlation between TfR ubiquitination and its iron-induced lysosomal degradation. The identification and characterization of a specific ubiquitin ligase for TfR is important in understanding the mechanism of iron homeostasis. Here, we show that membrane-associated RING-CH (MARCH) 8 ubiquitinates TfR and promotes its lysosomal degradation. Similar to other RING-type ubiquitin ligases, the RING-CH domain of MARCH8, which is located in the N-terminal cytoplasmic domain, is essential for the ubiquitination and downregulation of TfR. MARCH8 specifically recognizes the transmembrane domain of TfR and mediates ubiquitination of its cytoplasmic domain. In addition, the six-amino-acid sequence located in the C-terminal domain of MARCH8, which is highly conserved among different species, is required for the downregulation of TfR. Finally, and most importantly, TfR expression was markedly increased by siRNA-mediated knockdown of endogenous MARCH8. These findings demonstrate that the endogenous level of MARCH8 regulates TfR protein turnover through the downregulation and ubiquitination of TfR.

Sites of action of HIV-1 Vpu in BST-2/tetherin downregulation.

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of human immunodeficiency virus type 1 (HIV-1) by directly cross-linking virions to the membrane of infected cells. This antiviral effect is counteracted by the HIV-1 accessory protein viral protein U (Vpu) through mechanisms that remain unclear. Accumulating evidence suggests that Vpu antagonizes BST-2 by removing it from the plasma membrane; however, neither the cellular sites of interaction nor the effector mechanisms that result in the downregulation of BST-2 cell-surface expression have been fully determined. Based on current evidence regarding the subcellular localization of Vpu and BST-2 and the latter's trafficking defects induced by their interaction, three models have been proposed. In the first, Vpu is hypothesized to block the traffic of newly synthesized BST-2 towards the cell surface by retaining it in the biosynthetic/secretory compartment. The second model suggests that Vpu sequesters BST-2 within intracellular compartments corresponding to recycling endosomes and the trans-Golgi network by blocking its recycling after endocytosis. In the third model, we and others have proposed that Vpu directly internalizes BST-2 from the plasma membrane and induces its enhanced endolysosomal trafficking and degradation. As for its intracellular fate, the viral antagonism of BST-2 is likely dependent on the intracellular sequestration, or the proteasomal/lysosomal degradation of the restriction factor. This review summarizes the current advances in our understanding of the cellular pathways and sites of action of Vpu in the downregulation of cell-surface BST-2.

Structural Basis for the Antiviral Activity of BST-2/Tetherin and Its Viral Antagonism.

The interferon-inducible host restriction factor bone marrow stromal antigen 2 (BST-2/tetherin) blocks the release of HIV-1 and other enveloped viruses. In turn, these viruses have evolved specific antagonists to counteract this host antiviral molecule, such as the HIV-1 protein Vpu. BST-2 is a type II transmembrane protein with an unusual topology consisting of an N-terminal cytoplasmic tail (CT) followed by a single transmembrane (TM) domain, a coiled-coil extracellular (EC) domain, and a glycosylphosphatidylinositol (GPI) anchor at the C terminus. We and others showed that BST-2 restricts enveloped virus release by bridging the host and virion membranes with its two opposing membrane anchors and that deletion of either one completely abrogates antiviral activity. The EC domain also shows conserved structural properties that are required for antiviral function. It contains several destabilizing amino acids that confer the molecule with conformational flexibility to sustain the protein's function as a virion tether, and three conserved cysteine residues that mediate homodimerization of BST-2, as well as acting as a molecular ruler that separates the membrane anchors. Conversely, the efficient release of virions is promoted by the HIV-1 Vpu protein and other viral antagonists. Our group and others provided evidence from mutational analyses indicating that Vpu antagonism of BST-2-mediated viral restriction requires a highly specific interaction of their mutual TM domains. This interpretation is further supported and expanded by the findings of the latest structural modeling studies showing that critical amino acids in a conserved helical face of these TM domains are required for Vpu-BST-2 interaction and antagonism. In this review, we summarize the current advances in our understanding of the structural basis for BST-2 antiviral function as well as BST-2-specific viral antagonism.

Differential anti-APOBEC3G activity of HIV-1 Vif proteins derived from different subtypes.

Antiretroviral cytidine deaminase APOBEC3G, which is abundantly expressed in peripheral blood lymphocytes and macrophages, strongly protects these cells against HIV-1 infection. The HIV-1 Vif protein overcomes this antiviral effect by enhancing proteasome-mediated APOBEC3G degradation and is key for maintaining viral infectivity. The 579-bp-long vif gene displays high genetic diversity among HIV-1 subtypes. Therefore, it is intriguing to address whether Vif proteins derived from different subtypes differ in their viral defense activity against APOBEC3G. Expression plasmids encoding Vif proteins derived from subtypes A, B, C, CRF01_AE, and CRF02_AG isolates were created, and their anti-APOBEC3G activities were compared. Viruses produced from cells expressing APOBEC3G and Vif proteins from different subtypes showed relatively different viral infectivities. Notably, subtype C-derived Vif proteins tested had the highest activity against APOBEC3G that was ascribed to its increased binding activity, for which the N-terminal domain of the Vif protein sequences was responsible. These results suggest that the biological differences of Vif proteins belonging to different subtypes might affect viral fitness and quasispecies in vivo.

Direct internalization of cell-surface BST-2/tetherin by the HIV-1 accessory protein Vpu.

The host transmembrane protein BST-2/tetherin is a powerful antiviral factor that blocks the production of enveloped viruses. The HIV-1 accessory protein Vpu inhibits the antiviral activity of BST-2; however, the degradation pathway by which Vpu downregulates BST-2 from the cell surface and the actual subcellular location where Vpu targets BST-2 for downregulation remain controversial. Whereas one study showed that Vpu acts on constitutively endocytosed BST-2, we recently reported that Vpu can internalize BST-2 from the cell surface. Because the evidence for this conclusion was derived from indirect results, we present direct evidence in this study using an antibody internalization assay with an endocytosis-defective mutant of BST-2. The internalization of the BST-2 protein into cells coexpressing wild-type Vpu was observed when the cells were preincubated with antibodies against BST-2 at 37 degrees C, but not at 4 degrees C, for 10 min. These results strongly support our previous finding that continuously expressed de novo BST-2 at the cell surface is internalized by functional Vpu protein.

Combined analysis of cell growth and apoptosis-regulating proteins in HPVs associated anogenital tumors.

BACKGROUND: The clinical course of human papillomavirus (HPV) associated with Bowenoid papulosis and condyloma acuminatum of anogenital tumors are still unknown. Here we evaluated molecules that are relevant to cellular proliferation and regulation of apoptosis in HPV associated anogenital tumors. METHODS: We investigated the levels of telomerase activity, and inhibitor of apoptosis proteins (IAPs) family (c-IAP1, c-IAP2, XIAP) and c-Myc mRNA expression levels in 20 specimens of Bowenoid papulosis and 36 specimens of condyloma acuminatum in anogenital areas. Overall, phosphorylated (p-) AKT, p-ribosomal protein S6 (S6) and p-4E-binding protein 1 (4EBP1) expression levels were examined by immunohistochemistry in anogenital tumors both with and without positive telomerase activity. RESULTS: Positive telomerase activity was detected in 41.7% of Bowenoid papulosis and 27.3% of condyloma acuminatum compared to normal skin (p < 0.001). In contrast, the expression levels of Bowenoid papulosis indicated that c-IAP1, c-IAP2 and XIAP mRNA were significantly upregulated compared to those in both condyloma acuminatum samples (p < 0.001, p < 0.001, p = 0.022, respectively) and normal skin (p < 0.001, p = 0.002, p = 0.034, respectively). Overall, 30% of Bowenoid papulosis with high risk HPV strongly promoted IAPs family and c-Myc but condyloma acuminatum did not significantly activate those genes. Immunohistochemically, p-Akt and p-S6 expressions were associated with positive telomerase activity but not with p-4EBP1 expression. CONCLUSION: Combined analysis of the IAPs family, c-Myc mRNA expression, telomerase activity levels and p-Akt/p-S6 expressions may provide clinically relevant molecular markers in HPV associated anogenital tumors.

HIV-1 accessory protein Vpu internalizes cell-surface BST-2/tetherin through transmembrane interactions leading to lysosomes.

Bone marrow stromal antigen 2 (BST-2, also known as tetherin) is a recently identified interferon-inducible host restriction factor that can block the production of enveloped viruses by trapping virus particles at the cell surface. This antiviral effect is counteracted by the human immunodeficiency virus type 1 (HIV-1) accessory protein viral protein U (Vpu). Here we show that HIV-1 Vpu physically interacts with BST-2 through their mutual transmembrane domains and leads to the degradation of this host factor via a lysosomal, not proteasomal, pathway. The degradation is partially controlled by a cellular protein, beta-transducin repeat-containing protein (betaTrCP), which is known to be required for the Vpu-induced degradation of CD4. Importantly, targeting of BST-2 by Vpu occurs at the plasma membrane followed by the active internalization of this host protein by Vpu independently of constitutive endocytosis. Thus, the primary site of action of Vpu is the plasma membrane, where Vpu targets and internalizes cell-surface BST-2 through transmembrane interactions, leading to lysosomal degradation, partially in a betaTrCP-dependent manner. Also, we propose the following configuration of BST-2 in tethering virions to the cell surface; each of the dimerized BST-2 molecules acts as a bridge between viral and cell membranes.

Spectroscopic characterization of human immunodeficiency virus type-1-infected plasma by principal component analysis and soft independent modeling of class analogy of visible and near-infrared spectra.

Previous research has shown that it may be possible to diagnose infections of human immunodeficiency virus type-1 (HIV-1) using plasma by a partial least squares regression analysis of visible and near-infrared (Vis-NIR) spectra. In this study, the features of plasma in HIV-1-infected and healthy individuals were further investigated by Vis-NIR spectroscopy using principal component analysis (PCA) and soft independent modeling of class analogy (SIMCA). Although the mean Vis-NIR spectra of 33 HIV-1-infected individuals and 15 healthy donors showed only slight differences, the two groups were respectively distinguished using a score plot of the first versus second or second versus third principal components, and by a Coomans plot. The PCA loadings were generally consistent with the discriminating power of the SIMCA, indicating specific changes in Vis-NIR spectra after HIV-1-infection. The specific pattern possibly indicates ROH and RNH2, which may constitute specific features of components in HIV-1-infected plasma.

Superinfection of defective human immunodeficiency virus type 1 with different subtypes of wild-type virus efficiently produces infectious variants with the initial viral phenotypes by complementation followed by recombination.

Superinfection rates of human immunodeficiency virus type 1 (HIV-1) have increasingly been leading to more variation in HIV-1, as evidenced by the emergence of circulating recombinant forms (CRFs). We recently reported complementation in a persistently replication-defective subtype B-infected cell clone, L-2, by superinfection with CRF15_01B. The L-2 cells continuously produce immature particles due to a one-base insertion at pol protease. Proviruses in the superinfected cells carried both subtypes and produced particles with a mature morphology. In this study, we examined possible recombination following complementation to generate replication-competent variants by using three cell clones prepared from superinfected L-2 cells. The individual clones predominantly expressed the initial subtype B-derived mature Gag proteins. However, the viral particles carried both subtype B with the mutation and wild-type CRF15_01B at pol, suggesting the generation of virions with heterozygous RNAs. Interestingly, with cell-free passages of the progeny, defective particles disappeared, and were replaced with heterogeneous recombinants in the pol region with sequences derived from CRF15_01B that expressed subtype B phenotype. Thus, even a defective form of persistent HIV-1 can become replication-competent through superinfection-mediated complementation followed by recombination. These findings suggest the significance of long-lived infected cells as recipients for superinfection.

Inhibitory function of adapter-related protein complex 2 alpha 1 subunit in the process of nuclear translocation of human immunodeficiency virus type 1 genome.

The transfection of human cells with siRNA against adapter-related protein complex 2 alpha 1 subunit (AP2alpha) was revealed to significantly up-regulate the replication of human immunodeficiency virus type 1 (HIV-1). This effect was confirmed by cell infection with vesicular stomatitis virus G protein-pseudotyped HIV-1 as well as CXCR4-tropic and CCR5-tropic HIV-1. Viral adsorption, viral entry and reverse transcription processes were not affected by cell transfection with siRNA against AP2alpha. In contrast, viral nuclear translocation as well as the integration process was significantly up-regulated in cells transfected with siRNA against AP2alpha. Confocal fluorescence microscopy revealed that a subpopulation of AP2alpha was not only localized in the cytoplasm but was also partly co-localized with lamin B, importin beta and Nup153, implying that AP2alpha negatively regulates HIV-1 replication in the process of nuclear translocation of viral DNA in the cytoplasm or the perinuclear region. We propose that AP2alpha may be a novel target for disrupting HIV-1 replication in the early stage of the viral life cycle.

The RING finger ubiquitin ligase RNF125/TRAC-1 down-modulates HIV-1 replication in primary human peripheral blood mononuclear cells.

CXCR4-using HIV-1 was previously shown to replicate more efficiently in a healthy donor-derived CD4(+) CD38(+) than in a CD4(+) CD38(-) T-cell subset after stimulation with interleukin (IL)-4. Here, we identified 3 cellular genes, which were expressed to a higher level in an IL-4-stimulated CD38(-) subset. One of the 3 genes, RNF125/TRAC-1, was involved in the down-regulation of HIV-1 replication not only in cell lines, but also in peripheral blood mononuclear cells. RNF125/TRAC-1 bears the RING finger domain, important for E3 ubiquitin protein ligase. Mutations in this domain of RNF125/TRAC-1 led to the loss of HIV-1 down-modulatory activity, suggesting that E3 ligase activity is necessary. In addition, the results of Northern blotting and reporter gene analysis indicated that RNF125/TRAC-1 function occurs at the viral transcription step. These results suggest that RNF125/TRAC-1 could function to recruit host factor(s) controlling HIV-1 transcription to the ubiquitin-proteasome pathway.

Aberrant life cycle of human immunodeficiency virus type 1 CRF15_01B-like clinical isolates from Thailand in human CD4+ T-cell lines.

Human immunodeficiency virus type 1 (HIV-1) is separated into several subtypes and circulating recombinant forms (CRFs). Here, infections of 4 clinical isolates (0-47-1, CU98-26, CU98-28, and CU98-31) from Thailand were examined in human CD4(+) T-cell lines, MT-4 and MOLT-4. The CU98-26 isolates in both cells and 0-47-1 in MT-4 established chronic infections, as in control 2 subtype B isolates from Japan, while 0-47-1 in MOLT-4 caused a latent infection. In contrast, CU98-28 and CU98-31 established aberrant infections in both cells. Integrated provirus was detected in all the chronic infections, including 0-47-1 in both cells. In contrast, extrachromosomal circular forms of HIV-1 DNA were detected in CU98-28- and CU98-31-infected cells, whereas the amount of the integrated form was below the limit of detection. Interestingly, phylogenetic trees and sequencing revealed that all the Thai isolates, except 0-47-1, displayed CRF15_01B-like mosaic structures of CRF01_AE with subtype B-like sequences in several regions that were apparently different from those of the inocula in peripheral blood mononuclear cells. Thus, in the infections of most of the above Thai isolates it was suggested that a minor population with mosaic patterns having multiple breakpoints between CRF01_AE and subtype B in the inocula could be selected by the T-cell lines.

Superinfection of human immunodeficiency virus type 1 (HIV-1) to cell clone persistently infected with defective virus induces production of highly cytopathogenic HIV-1.

Superinfection with human immunodeficiency virus type 1 (HIV-1) in human subjects, defined as reinfection with a heterologous strain of HIV-1, has become a topic of great interest. To illustrate the significance of this occurrence, we performed HIV-1 superinfection of L-2 cells, which were isolated from MT-4 cells persistently infected with subtype B HIV-1 as a cell clone continuously producing defective HIV-1 particles. L-2 cells carrying provirus with a one-base insertion in the pol protease were superinfected with HIV-1 derived from primary isolates of subtype B or CRF01_AE. The kinetics of the superinfection in L-2 were very slow compared with those of primary infections in MT-4. Interestingly, L-2 shifted after superinfection to become a producer of highly cytopathogenic HIV-1. Molecular characterization revealed that superinfection occurred in only about 10% of the CRF01_AE-superinfected L-2, which carried provirus of both subtypes and produced viral particles containing genomic RNA of both subtypes. Surprisingly, such cytopathogenic HIV-1 showed predominantly the original subtype B phenotype. Thus, the mechanism of the production of cytopathic HIV-1 seemed to be mediated by trans complementation with pol products of superinfected CRF01_AE. These findings suggest the significance of long-lived infected cells as recipients for superinfection that could result in the generation of new HIV-1 variants with high virulence in patients who are off therapy or do not adhere to treatment, and may indicate the need for precautions against such superinfection.

A novel diagnostic method for human immunodeficiency virus type-1 in plasma by near-infrared spectroscopy.

Presently, the diagnosis of virus infections is based mainly on serological assays. Although polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA) have been increasingly used for the diagnosis of such viral infections, the risk of transfusion-transmitted blood-borne viruses remains. Furthermore, PCR and ELISA are expensive and time-consuming, and sometimes cause falsepositive or false-negative results. Therefore, a rapid, accurate and cost-effective diagnostic procedure is needed. We subjected plasma from individuals infected with human immunodeficiency virus type-1 (HIV-1), the causative agent of acquired immune deficiency syndrome (AIDS), as well as plasma from uninfected individuals as a control to near-infrared (NIR) spectroscopy, which may provide a rapid diagnostic method for HIV-1 infection without using any reagent. NIR spectra in the 600-1,000 nm region for plasma from pre-serologically HIV-1-infected individuals and healthy donors were subjected to partial least squares (PLS) regression analysis and leave-out cross-validation to develop a multivariate model to estimate the concentration of HIV-1. Simultaneously, the same plasma samples were examined for HIV-1 p24 by ELISA. The results obtained by the NIR spectroscopy model for HIV-1 yielded a good correlation with those obtained by the reference method (HIV-1 p24 ELISA). These results suggest that NIR spectroscopy using plasma could provide a rapid, accurate, cost-effective tool for large-scale diagnosis of HIV-1 infection.

Different susceptibility of human immunodeficiency virus type 1 to Env gp41-derived synthetic peptides corresponding to the C-terminal heptad repeat region.

Two functional domains, alpha-helical heptad repeat 1 (HR-1) and HR-2, located in the N-terminal and C-terminal regions of human immunodeficiency virus type 1 (HIV-1) Env gp41, respectively, play an important role in the fusion process. Synthetic 34-amino-acid peptide that contains the HR-2 region, named C34, has been shown to inhibit the HIV-1 fusion process. Here, we prepared six representative peptides (C34-B1, -B2, -A, -C1, -C2, and -E from subtypes B, A, C, and E, respectively) according to the sequences from the HIV sequence database of Los Alamos. All the C34 peptides had lower ability to inhibit the primary isolates (subtypes B and CRF01_AE) than subtype B laboratory strain LAI. On the other hand, the L-2 cell clone, isolated from persistently LAI-infected MT-4 cells (MT-4/LAI), showed unique C34 peptide sensitivities. L-2 virus has the same sequences at HR-1 and HR-2 regions as LAI, but showed higher syncytia formation activity than LAI. Interestingly, the sensitivity of L-2 was higher to C34-B2 and -A but slightly lower to C34-C1 at higher concentrations than MT-4/LAI, while C34-B1, -C2, and -E showed similar activity against both viruses. Thus, in addition to the sequences of the C34 peptide as well as of the HR-1 and HR-2 regions in target viruses used for fusion assays, the fusion inhibitory activities of C34 peptides seem to be affected by viral factor(s) other than the gp41 alpha-helical heptad repeats.

Higher frequency of premature stop codon mutations at vpu gene of human immunodeficiency virus type 1 CRF01_AE compared with those of other subtypes.

Our previous study demonstrated the anti-apoptosis function of the human immunodeficiency virus type 1 (HIV-1) vpu gene product in normal CD4+ T lymphocytes. In this study, using sequences obtained from the HIV sequence database, we compared vpu sequences from 184 preparations of various subtypes of HIV-1 from diverse geographical regions. Our analysis revealed that CRF01_AE isolates had premature stop codon mutations at the vpu gene at a much higher rate (36%) than other subtypes (0-9%). The premature stop codon mutations in vpu existed mostly at two amino acid residues: the methionine initiation codon and the boundary between the transmembrane (TM) and cytoplasmic domains. The mutations at the latter site were more often detected in CRF01_AE. The higher mutation rates at vpu in CRF01_AE were confirmed by sequence comparison of polymerase chain reaction products newly obtained directly from the DNA extracted from peripheral blood mononuclear cells (PBMCs), but not from the RNA from the plasma, in CRF01_AE- and subtype B-infected individuals. This finding may indicate the possibility that the more abundant population of HIV-1 CRF01_AE is able to induce apoptosis in CD4+ T lymphocytes than the populations of other subtypes.

Interleukin-4 up-regulates T-tropic human immunodeficiency virus type 1 transcription in primary CD4+ CD38+ T-lymphocyte subset.

The capacity of human immunodeficiency virus type 1 (HIV-1) to infect resting cells and to produce progeny particles may contribute significantly to its pathogenicity in vivo. We previously reported that primary culture of resting CD4(+) CD38(+) T-lymphocyte subset had higher production rate of CXCR4-using (X4) HIV-1 than CD4(+) CD38(-) subset. Interleukin (IL)-4 highly contributed to the up-regulation of the X4 virus production in the CD38(+) subset. Here, we show evidences that IL-4 treatment of both resting CD38(+) and CD38(-) subsets allowed the adsorption, entry, and integration of X4 virus at similar rates, while the following viral transcription rate was significantly lower in the CD38(-) than CD38(+) subset. Treatment of the CD38 subsets with IL-4 or phytohemagglutinin revealed no association of X4 virus replication ability in the subsets with classic T-cell activation or proliferation. Interestingly, the activator protein (AP)-1 was significantly activated in the CD38(+) subset after IL-4 treatment, while both nuclear factor (NF)-kappaB and signal transducers and activator of transcription (STAT)-6 were activated in the IL-4-treated CD38(-) and CD38(-) subsets at similar levels. Thus, IL-4-dependent X4 HIV-1 transcription occurs efficiently in the CD38(+) but not CD38(-) subset of CD4(+) population and AP-1 could play a significant role on viral transcription, leading to the up-regulated X4 virus production in the CD38(+) subset.

Enhancement of human immunodeficiency virus type 1 infectivity by replacing the region including Env derived from defective particles with an ability to form particle-mediated syncytia in CD4+T cells.

The infection and subsequent replication rates of human immunodeficiency virus type 1 (HIV-1) affect the pathogenicity. The initial stage of HIV-1 infection is largely regulated by viral envelope sequence. We previously reported that the defective doughnut-shaped particles produced from a persistently infected cell clone, named L-2, obtained from human CD4+ T-cell line MT-4 that was persistently infected with HIV-1 LAI strain, efficiently form particle-mediated syncytia with uninfected human CD4+ T-cell line, MOLT-4. Here, we prepared a molecular clone (pL2) containing the L-2 provirus to characterize the viral genetic region contributing to this activity to form particle-mediated syncytia. Several recombinants were constructed with pNL4-3 by replacing the pL2-derived region including full-length env. Characterization of the particles obtained by transfection with these recombinant clones confirmed that pL2-derived env carried the particle-mediated syncytia formation activity. It is noteworthy that the pL2-derived env region could also contribute to enhancement of infectivity in CD4+ T-cell lines as well as primary peripheral blood mononuclear cells (PBMCs). Thus, the HIV-1 particle-mediated syncytium formation activity could also contribute to the enhancement of HIV-1 infectivity.